CN108486107A - Novel mRNA based on bimolecular fluorescence complementary and circular rna labeling method - Google Patents
Novel mRNA based on bimolecular fluorescence complementary and circular rna labeling method Download PDFInfo
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Abstract
RNA molecule is a member important in intracellular hereditary information transmission system.The method of existing labeled RNA can not be provided simultaneously with living cells, without intrusion, high-spatial and temporal resolution, unimolecule the features such as, the method that needs to develop new label and track RNA.The present invention discloses a kind of labeling method based on the novel mRNA and circular rna from connection label bimolecular fluorescence complementary.In conjunction with RNA aptamer system, it is inserted into aptamer in the suitable position of RNA, by the binding protein of two part difference fusion rna aptamers of connection label certainly.When fusion protein unbound rna, RNA molecule does not fluoresce；When fusion protein combination RNA, RNA molecule sends out fluorescence, can reduce the fluorescence background of intracellular rna label in this way.The novel RNA labeling methods from connection label bimolecular fluorescence complementary can be used for the noiseless single molecular imaging observation of long time-histories and tracking of the mRNA and circular rna of living cells and fixed cell, have boundless application prospect in cell biology.
Description
Technical field
The present invention relates to a kind of new intracellular mRNAs and circular rna labeling method, and its in cell biology
Using.Belong to cell biology.
Background technology
Ribonucleic acid (RNA) molecule is the biography for being present in biological cell and the hereditary information in fractionated viral, viroid
Pass body.One ribonucleotide monomer molecule is made of a molecule phosphoric acid, ribose and base.The base master of ribonucleotide acid monomers
There are 4 kinds, i.e. adenine (A), guanine (G), cytimidine (C), uracil (U).RNA is by ribonucleotide through di-phosphate ester
Long chain high molecular polymer made of key condensation.Central dogma points out that DNA forms RNA by transcription, and RNA translates into albumen
Matter functions.RNA is using a chain of DNA as template, and with base pair complementarity principle, one for transcribing and being formed is single-stranded,
Major function is the transfer for realizing hereditary information from DNA to protein, is the bridge in hereditary information transmittance process.RNA types
It is various, including but not limited to mRNA (mRNA), rRNA (rRNA) and transfer RNA (tRNA).Discovered in recent years
Other number of types of RNA also play very important role, including long non-coding RNA in the vital movement of cell,
ERNA, circular rna etc..
In recent decades, the progress of microscope and RNA labelling techniques makes it possible the imaging of RNA molecule.In fixation
Us can be helped to obtain the information such as quantity, position, the dynamic of RNA molecule the direct label of RNA molecule in cell and living cells,
The dynamics for providing RNA metabolic processes, includes transcription, transhipment, modification, translation, the degradation etc. of RNA.The most common label sides RNA
Method is the fluorescence in situ hybridization for RNA sequence in fixed cell, this is the goldstandard method for positioning RNA molecule.One
The probe of up to tens complementary pairings is designed on RNA can increase signal, improve signal-to-noise ratio, can improve the positioning of RNA
To single molecules level.Fluorescence in situ hybridization also allows polychrome is high-throughput to mark positioning multiple types RNA simultaneously simultaneously.But fluorescence is former
The disadvantage of position hybridization is cannot to be compatible with living cells, needs the cells are fixed to punch and the probe of fluorescent marker could be sent into carefully
In born of the same parents, so can only obtain the location information of RNA, and lack multidate information.And the RNA imaging techniques in living cells can provide
The real time data of RNA Expression processes.The labeling method of living cells includes mainly molecular beacon, and RNA aptamer system can be compiled
The RNA collected identifies albumen.Molecular beacon is usually short DNA molecular, and both ends respectively connect a fluorescent dye groups and base is quenched
Group.When being not joined on RNA, the structure of pairing can be formed inside DNA molecular and so that fluorescent dye is quenched and does not shine.It is attached to
Internal structure is opened when on RNA, and RNA is made to shine.However, these probes cannot independently enter living cells, need to use micro- note
Penetrating equal invasive methods makes probe enter cell, this will generate certain injury to the normal physiological activity of cell.RNA is adapted to
Subsystem be RNA to be marked noncoding region be inserted into one section of RNA aptamer, co-express RNA aptamer binding protein or
The non-invasive marker that RNA may be implemented in permeable membrane combination dye is added.After the noncoding region of RNA is inserted into multiple aptamer,
It can realize the unimolecule label of RNA.This method is that RNA marks most common method in current living cells.But it is inserted into
The metabolic process of itself that RNA can be influenced after multiple aptamers, hinders the transport of RNA.Based on editable rna binding protein mark
The principle of note RNA molecule is the sequence by adjusting protein or sgRNA, and the RNA of any label can be not added with to endogenous
It is marked.But this method cannot achieve unimolecule label, simultaneously because editable rna binding protein is generally bigger, it can
It can influence the transportational process of RNA.
In addition, there is also the RNA types that some are difficult label in cell.Wherein important one kind is circular rna.It is cyclic annular
RNA is a kind of non-volume for not having the ends 5' cap and the ends 3' polyadenylic acid tail and loop configuration is formed with covalent bond
Code RNA molecule.Circular rna is generated by variable sheer, sometimes even over as many as 10 times of their linear isomers.Circular rna
Largely it is present in the cytoplasm of eukaryocyte, but the circular rna in small part introne source is then present in nucleus, has
Certain tissue, sequential and disease specific.Different from traditional linear rna, circular RNA molecule is in closed circular structure, no
Easily degraded by exonuclease RNaseR, it is more more stable than linear rna, have well-conserved.Most of circular rnas are from outer
Aobvious son, small part are directly cyclized by introne and are formed.Part cyclic RNA molecule response element containing miRNA, may act as in competitiveness
Source RNA, is combined with miRNA, plays the role of miRNA sponges in cell, and then is released miRNA and made to the inhibition of its target gene
With raising the expression of target gene.Part cyclic RNA can also translate into protein, but most of is non-coding RNA.By
In each circular rna and its corresponding linear isomers be all by the same precursor RNA montage froing, therefore annular RNA with
Linear RNA and precursor RNA share partial nucleotide sequence, it is difficult to distinguish three for the specific marker of sequence.
In conclusion need to develop at present may be implemented in living cells without it is invasive, on RNA itself influence it is smaller,
The novel RNA labeling methods of single-molecule resolution may be implemented.The present invention uses mutual based on the bimolecular fluorescence from connection label
The technology of benefit has developed the labelling technique of mRNA and circular rna in new living cells in conjunction with RNA aptamer system.Believing
The 5' non-translational regions end of RNA is set to be inserted into concatenated RNA aptamer MS2 and PP7, the two is at a distance of 20 bases.In suitable position
Two parts will be split into from connection label H aloTag by putting, HaloTag N and HaloTag C, difference fusion rna aptamer
Binding protein tdPCP and tdMCP.It does not fluoresce when the HaloTag protein fusions of division are without interaction protein, when two eggs
When having direct interaction in vain or relying on other holders close to each other, two sections of division can be made to connect label spatially phase certainly
It is mutually close, complete functional connection label certainly is formed, fluorescence can be sent out with combination dye.As HaloTag N-tdPCP and
When tdMCP-HaloTag C do not combine the aptamer on mRNA, messenger RNA molecule does not fluoresce, to reduce into the cell
Background.When aptamer on HaloTag N-tdPCP and tdMCP-HaloTag C combination mRNAs, mRNA
Molecule sends out fluorescence.The fluorescence background of intracellular rna label can be reduced in this way.Meanwhile because dye molecule brightness is high, courier
In RNA molecule unimolecule label can be carried out to messenger RNA molecule only in conjunction with a dye molecule.
And the labeling method of circular rna is similar with the label of mRNA but is slightly different.Before corresponding to circular rna
RNA aptamer MS2 and PP7 are inserted between the acceptor splicing site and donor splicing site of body mRNA respectively, makes the two after reversed cyclization
At a distance of 20 nucleotide.Inverted repeats is inserted into the upstream and downstream introne of acceptor splicing site and donor splicing site can make RNA
The higher structure of pairing is formed, acceptor splicing site and donor splicing site are close to each other, it is easier to reverse splicing occur.It is cut when forward direction occurs
When connecing, MS2 and PP7 are located remotely from each other, can not be spatially close to each other, in conjunction with HaloTag N-tdPCP and tdMCP- thereon
HaloTag C cannot be complementarily shaped to a complete molecule, not fluoresce.When reverse splicing occurs, MS2 and PP7 are in space
It is upper close to each other, it can be complementarily shaped to one complete point in conjunction with HaloTag N-tdPCP and tdMCP-HaloTag C thereon
Son fluoresces.The corresponding circular rna of some precursor RNA specifically can be only marked with this method without marking
Linear rna corresponding to it.It, can only in conjunction with a dye molecule on circular RNA molecule meanwhile because dye molecule brightness is high
To carry out unimolecule label to circular RNA molecule.
And used labeling method is organic dyestuff, is different from fluorescin, organic dyestuff is in an excitation process
In can release more photons, brightness higher.Organic dyestuff is more stable simultaneously, can resist photobleaching to a certain extent, can be with
The observation longer time.
In conclusion provided by the present invention based on RNA in the novel cell of the bimolecular fluorescence complementary of connection label certainly
The noiseless single molecular imaging of long time-histories that labeling method can be used for the mRNA and circular rna of living cells and fixed cell is seen
It surveys and tracks.There is boundless application prospect (Figure of description 1) in cell biology.
Invention content
The technical purpose of the present invention is developed based on RNA marks in the novel cell of the bimolecular fluorescence complementary of connection label certainly
The purpose of label and tracking intracellular mRNA and circular rna of the long-time without background in living cells may be implemented in note method,
To solve the problems, such as in living cells without invasive, influence is small, single molecules level marks and tracking RNA.
The present invention discloses a kind of based on mRNA label side in the novel cell of the bimolecular fluorescence complementary of connection label certainly
Method merges aptamer binding protein from connection label protein using division, realizes the courier for being inserted into aptamer in noncoding region
The label of RNA is imaged.It does not fluoresce when fusion protein does not combine messenger RNA molecule, is sent out when it combines messenger RNA molecule
Fluorescence, to realize the stable labelling of the RNA molecule of unstressed configuration background and the imaging tracking of long time-histories in living cells, to provide courier
Multidate information in RNA life cycles.
The present invention discloses a kind of label sides novel cell inner annular RNA based on the bimolecular fluorescence complementary from connection label
Method.Be inserted into respectively between the acceptor splicing site and donor splicing site of precursor mRNA corresponding to circular rna RNA aptamer MS2 and
PP7 makes the two after reverse splicing cyclization at a distance of 20 nucleotide.Using division aptamer knot is merged from connection label protein
Hop protein realizes the label imaging of circular rna.It does not fluoresce when fusion protein does not combine circular RNA molecule, in its coupling collar
Fluorescence is sent out when shape RNA molecule, is not fluoresced when being attached at it on linear RNA, unstressed configuration is carried on the back in living cells to realize
The stable labelling of the annular RNA molecule of scape and the imaging tracking of long time-histories, help the formation and the transporting mechanism that understand circular rna.
MRNA and annular in novel cell disclosed by the invention based on the bimolecular fluorescence complementary for connecting label certainly
RNA labeling methods, used system can be living cells, can also be fixed cell.It can be with when being used in living cells
The subcellular localization of intracellular mRNA and annular RNA is observed, molecule dynamic transportational process is tracked, counts absolute molecular quantity
Deng.When in fixed cell in use, can be used for observing mRNA and annular RNA subcellular localizations, statistics absolute molecular number
Amount etc..
The present invention discloses a kind of based on mRNA and annular in the novel cell of the bimolecular fluorescence complementary of connection label certainly
In specific implementation process be inserted into adaptation can be adjusted flexibly according to the RNA molecule type marked in RNA labeling methods
Sub- position and conformation.In the label application of mRNA, the ends 5' noncoding region can be inserted in aptamer, can also be inserted into the ends 3'
Noncoding region.In order to not influence the translation process of mRNA, aptamer is generally inserted into the ends the 3' noncoding region of mRNA.
In the label application of annular RNA, limitation is compared in the insertion position of aptamer, is generally placed upon the donor splicing site and acceptor splicing site of cyclization
Between, and two aptamers will be separately inserted.
It is disclosed in this invention it is a kind of based on from connection label bimolecular fluorescence complementary novel cell in mRNA and
Annular RNA labeling methods, include the following steps：
(1) corresponding adaptor sequence is inserted into the suitable position of mRNA to be marked or annular RNA first.
(2) it determines from the Division site for connecting label protein, two sections of albumen of division is gone out by PCR amplification, it is distinguished
It is fused on aptamer binding protein, is connected into the carrier with expression cassette, is built into plasmid.
(3) by constructed plasmid co-transfection mammalian cell.
(4) dyestuff is added, observes the structure and dynamic of cell messenger RNA or annular RNA under the microscope.
Description of the drawings
MRNAs and circular rna labeling method schematic diagram of the Fig. 1 from connection label bimolecular fluorescence complementary.Upper figure：Courier
The labeling method of RNA.It is inserted into concatenated RNA aptamer MS2 and PP7 in the ends 3' of mRNA, the two is at a distance of 20 bases.
Two parts will be split into from connection label H aloTag in suitable site, HaloTag N and HaloTag C are merged respectively
The binding protein tdPCP and tdMCP of RNA aptamer.Do not send out glimmering when the HaloTag protein fusions of division are without interaction protein
Light can be such that two sections of division connect certainly when two albumen have direct interaction or rely on other holders close to each other
Label is spatially close to each other, forms complete functional connection label certainly, can send out fluorescence with combination dye.When
When HaloTag N-tdPCP and tdMCP-HaloTag C do not combine the aptamer on mRNA, messenger RNA molecule is not sent out glimmering
Light.When aptamer on HaloTag N-tdPCP and tdMCP-HaloTag C combination mRNAs, mRNA point
Son sends out fluorescence.The fluorescence background of RNA labels can be reduced in this way.Meanwhile because dye molecule brightness is high, messenger RNA molecule
On only in conjunction with a dye molecule can to messenger RNA molecule carry out unimolecule label.Figure below：The labeling method of circular rna.
Be inserted into respectively between the acceptor splicing site and donor splicing site of precursor mRNA corresponding to circular rna RNA aptamer MS2 and
PP7 makes the two after reversed cyclization at a distance of 20 nucleotide.It is inserted into the upstream and downstream introne of acceptor splicing site and donor splicing site
Inverted repeats can make RNA form the higher structure of pairing, and acceptor splicing site and donor splicing site are close to each other, it is easier to occur
Reverse splicing.When positive montage occurs, MS2 and PP7 are located remotely from each other, can not be spatially close to each other, in conjunction with thereon
HaloTag N-tdPCP and tdMCP-HaloTag C cannot be complementarily shaped to a complete molecule, not fluoresce.It is anti-when occurring
To when montage, MS2 and PP7 are spatially close to each other, in conjunction with HaloTag N-tdPCP and tdMCP-HaloTag C thereon
It can be complementarily shaped to a complete molecule, fluoresced.Some precursor specifically can be only marked with this method
The corresponding circular rnas of RNA are without marking the linear rna corresponding to it.Meanwhile because dye molecule brightness is high, circular RNA molecule
On only in conjunction with a dye molecule can to circular RNA molecule carry out unimolecule label.
Fig. 2 marks unimolecule mRNA and other methods to mark courier based on the bimolecular fluorescence complementary from connection label
RNA is compared.(a) RNA is inserted into tag size comparison.Using based on the bimolecular fluorescence complementary label unimolecule letter from connection label
RNA is set only to need to be inserted into a MS2 and PP7 at the ends 3' of the protein coding gene of target RNA, size is only 120 cores
Thuja acid.24 are generally required using the identical RNA aptamer of series multiple or the different aptamer system of series multiple to fit
Gamete, length reach 1500 nucleotide.For length is only the mRNA of several thousand bases, new labeling method pair
The influence of RNA itself is smaller.(b) rna binding protein size compares.Using based on the bimolecular fluorescence complementary mark from connection label
The unimolecule messenger RNA molecule of note thereon only combine HaloTag N-tdPCP and tdMCP-HaloTag a C, label it is total
Molecular weight is 96.1 kilodaltons.And utilize the identical RNA aptamer of series multiple or series multiple different with subsystem
The unimolecule RNA of system label needs the labelled protein in conjunction with 1153.2 kilodaltons thereon.In conclusion from tag size is inserted into
For two angles of binding protein size, unimolecule mRNA is marked based on the bimolecular fluorescence complementary from connection label
Method wants Billy's identical RNA aptamer of series multiple or the different aptamer system of series multiple to mRNA sheet
Figure rings smaller.
Fig. 3 marks unimolecule mRNA based on the bimolecular fluorescence complementary from connection label.(a) fluorescence of unimolecule RNA
Image.In the aptamer MS2 and PP7 that the ends the 3' insert the distance of CFP genes is 20 bases, using based on pair from connection label
The method of molecular fluorescence complementary indicia unimolecule mRNA is marked.In human breast cancer cell MDA-MB-231 altogether
Transfect corresponding plasmid.The corresponding messenger RNA molecules of CFP are marked after dyestuff is added.CFP gene expression cyanic colours fluorescins,
Cell for the cell and untransfected of distinguishing transfection.In the cell of expression CFP protein, it can be detected in the channels 561nm
To corresponding mRNA single molecule signals.(b) single point of CFP is marked using based on the bimolecular fluorescence complementary from connection label
Sub- mRNA is an intracellular molecular motion track.(c) list of the identical RNA aptamer label CFP of series multiple is utilized
Molecule mRNA is an intracellular molecular motion track.(d) using based on the bimolecular fluorescence complementary mark from connection label
Remember the unimolecule mRNA of CFP and being averaged for the unimolecule mRNA that CFP is marked using the identical RNA aptamer of series multiple
Square displacement.(e) it marks the unimolecule mRNA of CFP using the bimolecular fluorescence complementary based on connection label certainly and utilizes more
Identical RNA aptamer of connecting again marks the diffusion coefficient histogram frequency distribution diagram of the unimolecule mRNA of CFP.
Fig. 4 utilizes the bimolecular fluorescence complementary from connection label to mark unimolecule circular rna.(a) the label side of circular rna
Method.(ZKSCAN1548-1047) difference between the acceptor splicing site and donor splicing site of precursor mRNA corresponding to circular rna
It is inserted into RNA aptamer MS2 and PP7, makes the two after reversed cyclization at a distance of 20 nucleotide.In acceptor splicing site and donor splicing site
It is inserted into inverted repeats in upstream and downstream introne.The sequence of inverted repeats, donor splicing site, acceptor splicing site, MS2 and PP7
All marked in figure.(b) the cell light field channel of circular rna label.(c) the cell fluorescence channel of circular rna label.It is white in figure
Color dashed lines labeled goes out the profile of cell, and white arrow indicates the single molecule signals of circular rna.
Specific implementation mode
The technical purpose of the present invention is to develop the novel fine intracellular messenger based on the bimolecular fluorescence complementary from connection label
RNA and annular RNA labeling methods, which is characterized in that the mRNA of unstressed configuration background and annular RNA in living cells may be implemented
The stable labelling of molecule and the imaging tracking of long time-histories, to provide the multidate information in mRNA and annular RNA life cycles.Under
Face is illustrated in connection with specific embodiments.It, now will be according to the present invention for the clearer method content for illustrating the present invention
Product and method are further summarized as follows, and involved experimental data, step or synthetic method etc. belong to this field
Routine techniques does not cause to limit to the protection domain of this patent.These specific embodiments are served only for illustrating the present invention, and
It is not used in and limits the scope of the invention.
Embodiment one：Intracellular mRNA is marked based on the bimolecular fluorescence complementary from connection label.With division used
HaloTag label CFP mRNA for illustrate：
(1) the positive anti-chain of sequence of MS2 and PP7, equal proportion mixing are directly synthesized, annealing is connected into the non-coding of the gene of CFP
The area ends 3' obtain carrier pcDNA3.1 (+)-CFP-MS2-PP7.
(2) corresponding DNA fragmentation is amplified from the plasmid containing tdMCP and tdPCP by PCR.In the primer of amplification
Upper addition restriction enzyme site.It recycles to obtain pure DNA pieces by DNA agarose gel electrophoresis and gel purification kit
Section, obtains tdMCP and tdPCP segments.Used PCR amplification system is as follows：
(3) tdMCP and tdPCP recycled upper step and eukaryotic expression vector pcDNA3.1 (+) is respectively using limitation
Enzyme double digestion is stayed overnight, and recycles to obtain pure DNA fragmentation by DNA agarose gel electrophoresis and gel purification kit.It is used
Digestion system it is as follows：
(4) carrier for having used identical digestion will be connected into respectively by the tdMCP and tdPCP of digestion recycling
PcDNA3.1 (+) obtains carrier pcDNA3.1 (+)-tdPCP and pcDNA3.1 (+)-tdMCP.Used linked system is such as
Under：
(5) corresponding HaloTag divisions segment is amplified from the plasmid containing complete HaloTag by PCR.Expanding
Restriction enzyme site is added on the primer of increasing.It recycles to obtain by DNA agarose gel electrophoresis and gel purification kit pure
DNA fragmentation, obtain HaloTag N58 and HaloTag C58.Used PCR amplification system is with reference to step (2).
(6) the HaloTag N58 and expression vector pcDNA3.1 (+)-tdPCP recycled upper step uses restriction enzyme double digestion
Connection.HaloTag C58 are connected with expression vector pcDNA3.1 (+)-tdMCP using restriction enzyme double digestion.Used enzyme
It cuts system and linked system respectively refers to step (3) and (4).Respectively obtain carrier pcDNA3.1 (+)-HaloTag N58-
TdPCP and pcDNA3.1 (+)-tdMCP-HaloTag C58.
(7) human breast cancer cell line MDA-MB-231 is digested using pancreatin, is planted in again according to the density of 50-60%
It is imaged in dedicated 35mm Petri-Dish, cultivates 24 hours.
(8) above-mentioned cell uses each 1 μ g carriers pcDNA3.1 (+)-CFP- of liposome Lipo-2000 cotransfections of 6mL
MS2-PP7, pcDNA3.1 (+)-HaloTag N58-tdPCP and pcDNA3.1 (+)-tdMCP-HaloTag C58.Transfection institute
Culture medium is the Opti-MEM without serum.It has transfected 5 hours, has sucked transfection liquid, changed fresh culture medium.Continue to train
It supports 24 hours.
(9) the bound substrates dyestuff JF549 of HaloTag is added in the cell culture medium of above-mentioned transfection, it is final concentration of
10nM.Continue culture 20 minutes in the incubator, to make cell be uniformly mixed with dyestuff.
(10) 1 × PBS cleanings cell is used three times, to remove responseless free dye.Finally changed after the completion of cleaning
It is upper to be free of phenol red culture medium.
(11) intracellular fluorescent brightness is observed using the high power total internal reflectance microscope with living cells culture apparatus.Choosing
The more suitable cell of expression quantity is selected, the single molecule experiments data of mRNA are acquired.Experimental result is shown in Figure of description 2 and 3.
Embodiment two：Based on the bimolecular fluorescence complementary label intracellular cyclic RNA from connection label.With division used
HaloTag label ZKSCAN1 (548-1047) circular rna for illustrate：
(1) the positive anti-chain of sequence of MS2 and PP7, equal proportion mixing are directly synthesized, annealing is connected into ZKSCAN1 (548-1047)
Between donor splicing site and receptor, carrier pcDNA3.1 (+)-CircRNA Mini Vector-MS2-ZKSCAN1 (548- are obtained
1047)-PP7。
(2) human breast cancer cell line MDA-MB-231 is digested using pancreatin, is planted in again according to the density of 50-60%
It is imaged in dedicated 35mm Petri-Dish, cultivates 24 hours.
(3) above-mentioned cell uses each 1 μ g carriers pcDNA3.1 (+)-CircRNA of liposome Lipo-2000 cotransfections of 6mL
Mini Vector-MS2-ZKSCAN1 (548-1047)-PP7, pcDNA3.1 (+)-HaloTag N58-tdPCP, and
pcDNA3.1(+)-tdMCP-HaloTag C58.Transfection culture medium used is the Opti-MEM without serum.It is small to have transfected 5
When, transfection liquid is sucked, fresh culture medium is changed.Continue culture 24 hours.
(9) the bound substrates dyestuff JF549 of HaloTag is added in the cell culture medium of above-mentioned transfection, it is final concentration of
10nM.Continue culture 20 minutes in the incubator, to make cell be uniformly mixed with dyestuff.
(10) 1 × PBS cleanings cell is used three times, to remove responseless free dye.Finally changed after the completion of cleaning
It is upper to be free of phenol red culture medium.
(11) intracellular fluorescent brightness is observed using the high power total internal reflectance microscope with living cells culture apparatus.Choosing
The more suitable cell of expression quantity is selected, the single molecule experiments data of circular rna are acquired.Experimental result is shown in Figure of description 4.
SEQUENCE LISTING
<110>Peking University
<120>Novel mRNA based on bimolecular fluorescence complementary and circular rna labeling method
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 357
<212> PRT
<213>Artificial sequence
<400> 1
Met Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val
1 5 10 15
Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp
20 25 30
Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val
35 40 45
Trp Arg Asn Ile Ile Pro His Val Ala Pro Gly Ser Gly Gly Ser Gly
50 55 60
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Phe
65 70 75 80
Pro Lys Lys Lys Arg Lys Val Gly Tyr Pro Asp Tyr Lys Asp Asp Asp
85 90 95
Asp Lys Ala Cys Ser Leu Ala Ser Lys Thr Ile Val Leu Ser Val Gly
100 105 110
Glu Ala Thr Arg Thr Leu Thr Glu Ile Gln Ser Thr Ala Asp Arg Gln
115 120 125
Ile Phe Glu Glu Lys Val Gly Pro Leu Val Gly Arg Leu Arg Leu Thr
130 135 140
Ala Ser Leu Arg Gln Asn Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu
145 150 155 160
Lys Leu Asp Gln Ala Asp Val Val Asp Ser Gly Leu Pro Lys Val Arg
165 170 175
Tyr Thr Gln Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr
180 185 190
Glu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala
195 200 205
Thr Ser Gln Val Glu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg
210 215 220
Ala Asp Pro Leu Ala Ser Lys Thr Ile Val Leu Ser Val Gly Glu Ala
225 230 235 240
Thr Arg Thr Leu Thr Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe
245 250 255
Glu Glu Lys Val Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser
260 265 270
Leu Arg Gln Asn Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu
275 280 285
Asp Gln Ala Asp Val Val Asp Ser Gly Leu Pro Lys Val Arg Tyr Thr
290 295 300
Gln Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr Glu Ala
305 310 315 320
Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala Thr Ser
325 330 335
Gln Val Glu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg Pro Lys
340 345 350
Lys Lys Arg Lys Val
355
<210> 2
<211> 530
<212> PRT
<213>Artificial sequence
<400> 2
Met Gly Pro Lys Lys Lys Arg Lys Val Gly Tyr Pro Tyr Asp Val Pro
1 5 10 15
Asp Tyr Ala Leu Glu Val Leu Phe Gln Gly Pro Leu Leu Leu Ile Asn
20 25 30
Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr Gly
35 40 45
Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu Trp
50 55 60
Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val
65 70 75 80
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val
85 90 95
Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
100 105 110
Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln
115 120 125
Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn
130 135 140
Ser Gly Ile Tyr Ala Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val
145 150 155 160
Asp Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala
165 170 175
Asn Gly Ile Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
180 185 190
Lys Val Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr
195 200 205
Thr Ile Lys Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn
210 215 220
Met Glu Leu Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu
225 230 235 240
Ile Val Lys Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro
245 250 255
Ser Ala Ile Ala Ala Asn Ser Gly Ile Tyr Ala Asp Gly Ser Gly Gly
260 265 270
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
275 280 285
Glu Phe Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys
290 295 300
Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe
305 310 315 320
Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val
325 330 335
Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn
340 345 350
Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile
355 360 365
Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala
370 375 380
Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val
385 390 395 400
Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu
405 410 415
Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg
420 425 430
Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro
435 440 445
Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln
450 455 460
Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile
465 470 475 480
Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys
485 490 495
Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro
500 505 510
Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile
515 520 525
Ser Gly
530
<210> 3
<211> 865
<212> RNA
<213>Artificial sequence
<400> 3
auggugagca agggcgagga gcuguucacc gggguggugc ccauccuggu cgagcuggac 60
ggcgacguaa acggccacaa guucagcgug uccggcgagg gcgagggcga ugccaccuac 120
ggcaagcuga cccugaaguu caucugcacc accggcaagc ugcccgugcc cuggcccacc 180
cucgugacca cccugaccug gggcgugcag ugcuucagcc gcuaccccga ccacaugaag 240
cagcacgacu ucuucaaguc cgccaugccc gaaggcuacg uccaggagcg caccaucuuc 300
uucaaggacg acggcaacua caagacccgc gccgagguga aguucgaggg cgacacccug 360
gugaaccgca ucgagcugaa gggcaucgac uucaaggagg acggcaacau ccuggggcac 420
aagcuggagu acaacuacau cagccacaac gucuauauca ccgccgacaa gcagaagaac 480
ggcaucaagg ccaacuucaa gauccgccac aacaucgagg acggcagcgu gcagcucgcc 540
gaccacuacc agcagaacac ccccaucggc gacggccccg ugcugcugcc cgacaaccac 600
uaccugagca cccaguccgc ccugagcaaa gaccccaacg agaagcgcga ucacaugguc 660
cugcuggagu ucgugaccgc cgccgggauc acucucggca uggacgagcu guacaagucc 720
ggacucagau cucgagcuca agcuucgaau ucuuuaauac gguacuuauu gccaagaaag 780
cacgagcauc agccgugccu ccaggucgaa ucuucaaacg agcagacgau auggcgucgc 840
ucgcuccagu auuccagggu ucauc 865
<210> 4
<211> 707
<212> RNA
<213>Artificial sequence
<400> 4
aaagugcuga gauuacaggc gugagccacc acccccggcc cacuuuuugu aaagguacgu 60
acuaaugacu uuuuuuuuau acuucaggau uucaaacgag cagacgauau ggcgucgcuc 120
gcgaauagua aagaaacaca ucauaaaacc ucccaggaca uaaaggugag cacagacccu 180
guuuggauca agucaguucc uggagccuga augaugacug cugaaucacg ggaagccacg 240
ggucuguccc cacaggcugc acaggagaag gaugguaucg uaauagugaa gguggaagag 300
gaagaugagg aagaccacau gugggggcag gauuccaccc uacaggacac gccuccucca 360
gacccagaga uauuccgcca acgcuucagg cgcuucuguu accagaacac uuuugggccc 420
cgagaggcuc ucagucggcu gaaggaacuu ugucaucagu ggcugcggcc agaaauaaac 480
accaaggaac agauccugga gcuucuggug cuagagcagu uucuuuccau ccugcccaag 540
gagcuccagg ucuggcugca ggaauaccgc cccgauagug gagaggaggc cgugacccuu 600
cuagaagacu uggagcuuga uugcacgagc aucagccgug ccgcggaggu aagaagcaag 660
gaaaagaauu aggcucggca cgguagcuca caccuguaau cccagca 707
Claims (9)
1. mRNA and annular RNA labeling methods in a kind of novel cell based on the bimolecular fluorescence complementary for connecting label certainly,
It is characterized in that, being inserted into corresponding adaptor sequence in the suitable position of mRNA to be marked or annular RNA, division is utilized
From connection tag fusion aptamer binding protein, to realize the mRNA of unstressed configuration background and annular RNA in living cells
Unimolecule marks and imaging.
2. according to the method described in claim 1, it is characterized in that, not sending out glimmering when fusion protein does not combine messenger RNA molecule
Light sends out fluorescence, to reduce imaging background when it combines messenger RNA molecule.
3. according to the method described in claim 1, it is characterized in that, not sending out glimmering when fusion protein does not combine circular RNA molecule
Light sends out fluorescence when it combines circular RNA molecule, does not fluoresce when being attached at it on linear RNA, to specificity mark
Note circular rna simultaneously reduces imaging background.
4. according to the method described in claim 1-3, which is characterized in that used system can be living cells, can also be
Fixed cell.
5. according to the method described in claim 1-3, a kind of novel cell based on the bimolecular fluorescence complementary from connection label
Interior mRNA and annular RNA labeling methods, which is characterized in that the used label of connection certainly can be HaloTag,
SnapTag, ClipTag etc..
6. according to the method described in claim 1-5, a kind of novel cell based on the bimolecular fluorescence complementary from connection label
Interior mRNA and annular RNA labeling methods, which is characterized in that used fluorescent dye is small molecule permeable membrane dyestuff.
7. according to the method described in claim 1-6, a kind of novel cell fibril framework label based on bimolecular fluorescence complementary
Method, which is characterized in that prepared by following steps：
It first is inserted into corresponding adaptor sequence in the suitable position of mRNA to be marked or annular RNA, then is determined from connection mark
The Division site for signing albumen builds connection label and the protein-bonded fusion protein of aptamer certainly of division, by constructed matter
Grain cotransfection mammalian cell is added dyestuff and observes intracellular mRNA or annular RNA structures and dynamic under the microscope.
8. method described in claim 1-7 is under normal operation in cell in the label and imaging of mRNA or annular RNA
Using.
9. the method described in claim 1-7 under the conditions of pathology or dosing in cell the label of mRNA or annular RNA and
Application in imaging.
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